Exploring Structure–Activity Relationships of Niclosamide-Based Colistin Potentiators in Colistin-Resistant Gram-Negative Bacteria

Colistin is primarily used as a last resort antibiotic against highly resistant Gram-negative bacteria (GNB). Rising rates of colistin resistance, however, may limit future use of this agent. The anthelmintic drug niclosamide has been shown to enhance colistin activity in combination therapy, but a detailed structure–activity relationship (SAR) for niclosamide against GNB has yet to be studied. A series of niclosamide analogs were synthesized to perform an SAR, leading to the discovery of a lead compound that displayed comparable colistin-potentiating activity to niclosamide with reduced cytotoxicity. Overall, this work provides important insights into synthetic strategies for the future development of new niclosamide derivatives and demonstrates that toxicity to mammalian cells can be reduced while maintaining colistin potentiation.


Introduction
The widespread and ever-increasing threat of antimicrobial-resistant (AMR) "superbugs" presents a significant danger to the healthcare system and to the entire world [1,2].Indeed, a substantial increase in infections caused by AMR Gram-negative bacteria (GNB) has led to the increase in usage of last resort antibiotics such as colistin.However, rising rates of colistin resistance have resulted in bacterial infections that are resistant to all currently used antibiotics, leading to the deaths of several patients [3].If no new treatments for these challenging infections are developed, humanity may be left with no effective agents with which to eradicate these "superbugs".
A significant barrier to the development of new antibiotics is the challenging and costly drug approval process [4].This combined with limited return on investment for treating rare AMR infections has stagnated the development of new antibiotics [5].One method to overcome this significant challenge is to repurpose existing FDA-approved drugs towards the fight against AMR bacteria [6].The already established toxicity and pharmacokinetic profiles of these drugs may streamline and potentially truncate the development and approval process for such agents, rendering a quick pathway for their much needed use in the clinic.An example of such is the anthelmintic drug niclosamide approved for the treatment of tapeworm infections, which has been shown to exhibit antibacterial activity against Gram-positive bacteria [7], inhibit quorum sensing in Gram-negative bacteria [8], and able to synergize with colistin against GNB [9] to combat the development of colistin resistance.Despite being a promising candidate for repurposing as a drug against AMR infections, niclosamide has notable limitations.In particular, niclosamide has an extremely poor bioavailability primarily due to its low solubility [10].Toxicity to mammalian cells has also been observed which has spurred interest in repurposing niclosamide as an anticancer agent [11] but is a significant hindrance for developing it as a selective and safe antibacterial agent.In order to further develop niclosamide as an adjuvant capable of augmenting colistin and overcome colistin resistance, new derivatives must be synthesized, and their structure-activity relationships need to be studied.Several publications have reported new niclosamide analogs for combating colistin resistance [12], as well as a detailed mechanistic understanding of niclosamide in combination with colistin [13].
The aim of this study is to synthesize derivatives of niclosamide with the goal of reducing toxicity to human cells while retaining synergy with colistin.Niclosamide is composed of a 5-chlorosalicylic acid ring linked to a 2-chloro-4-nitroaniline ring via an amide bond.The nitro group has been demonstrated to contribute to genotoxicity [14], as well as serving as a target for bacterial nitroreductases resulting in resistance to niclosamide [13].These factors, in addition to reports that structurally similar salicylanilides such as rafoxanide (Figure 1) which lack the nitro group can also synergize with colistin [15], led to us targeting this site for most of our modifications.Our initial synthetic goal was the replacement of the nitro group with an amide moiety, which would allow rapid synthesis of a small library of amide analogs incorporating various functional groups.We also explored attaching azide and alkyne functional groups to the niclosamide scaffold with the goal of producing a small library of triazole-containing compounds via copper-assisted azide-alkyne cycloaddition (CuACC) [16].
negative bacteria [8], and able to synergize with colistin against GNB [9] to combat the development of colistin resistance.
Despite being a promising candidate for repurposing as a drug against AMR infections, niclosamide has notable limitations.In particular, niclosamide has an extremely poor bioavailability primarily due to its low solubility [10].Toxicity to mammalian cells has also been observed which has spurred interest in repurposing niclosamide as an anticancer agent [11] but is a significant hindrance for developing it as a selective and safe antibacterial agent.In order to further develop niclosamide as an adjuvant capable of augmenting colistin and overcome colistin resistance, new derivatives must be synthesized, and their structure-activity relationships need to be studied.Several publications have reported new niclosamide analogs for combating colistin resistance [12], as well as a detailed mechanistic understanding of niclosamide in combination with colistin [13].
The aim of this study is to synthesize derivatives of niclosamide with the goal of reducing toxicity to human cells while retaining synergy with colistin.Niclosamide is composed of a 5-chlorosalicylic acid ring linked to a 2-chloro-4-nitroaniline ring via an amide bond.The nitro group has been demonstrated to contribute to genotoxicity [14], as well as serving as a target for bacterial nitroreductases resulting in resistance to niclosamide [13].These factors, in addition to reports that structurally similar salicylanilides such as rafoxanide (Figure 1) which lack the nitro group can also synergize with colistin [15], led to us targeting this site for most of our modifications.Our initial synthetic goal was the replacement of the nitro group with an amide moiety, which would allow rapid synthesis of a small library of amide analogs incorporating various functional groups.We also explored attaching azide and alkyne functional groups to the niclosamide scaffold with the goal of producing a small library of triazole-containing compounds via copper-assisted azide-alkyne cycloaddition (CuACC) [16].In this work, it was found that the nitro group in niclosamide could be replaced with a methyl ester, an azide, or (to a lesser extent) a primary amine while retaining synergy with colistin against colistin-resistant GNB.We also show that replacement of the nitro group can result in new derivatives of niclosamide with lower cytotoxicity against human cells.Overall, this work provides important insights towards future development and optimization of niclosamide for overcoming colistin resistance.In this work, it was found that the nitro group in niclosamide could be replaced with a methyl ester, an azide, or (to a lesser extent) a primary amine while retaining synergy with colistin against colistin-resistant GNB.We also show that replacement of the nitro group can result in new derivatives of niclosamide with lower cytotoxicity against human cells.Overall, this work provides important insights towards future development and optimization of niclosamide for overcoming colistin resistance.

Chemistry
The nitro group on niclosamide was chosen as an initial site for modification.We initially sought to synthesize two series of amide analogs, one of which linked the amide to the salicylanilide scaffold via the nitrogen and one that was linked via the carbonyl group.The first series of derivatives resulted from treating niclosamide with zinc dust in the presence of NH 4 Cl, yielding primary amine derivative 1.As an initial proof of concept, the amine was further acylated and alkylated to produce compounds 2 and 3 (Scheme 1).

Chemistry
The nitro group on niclosamide was chosen as an initial site for modification.We initially sought to synthesize two series of amide analogs, one of which linked the amide to the salicylanilide scaffold via the nitrogen and one that was linked via the carbonyl group.The first series of derivatives resulted from treating niclosamide with zinc dust in the presence of NH4Cl, yielding primary amine derivative 1.As an initial proof of concept, the amine was further acylated and alkylated to produce compounds 2 and 3 (Scheme 1).The other series of amide containing compounds was synthesized by coupling commercially available 5-chlorosalisylic acid with various aniline derivatives.A methyl ester derivative 5a was synthesized via PCl3 coupling.Ester hydrolysis with LiOH produced carboxylic acid analogue 6 which was intended to serve as a scaffold to produce a small library of amide analogues (Schemes 2 and 3).
Initial attempts to derivatize acid 6 via amide coupling proved unsuccessful with analysis revealing the phenol in niclosamide posing synthetic challenges.Two phenol protecting group strategies were developed to overcome this challenge: a methyl ether protecting group that could be deprotected using BBr3 and a benzyl protecting group that could be deprotected with catalytic hydrogenolysis.The role of the phenol functional group was also explored using intermediate 5b, analogous to 5a but with a methyl ether protected phenol.These protecting group strategies enabled the development of amide derivatives 7-12 which allowed for the introduction of a variety of functional groups (Scheme 3).The other series of amide containing compounds was synthesized by coupling commercially available 5-chlorosalisylic acid with various aniline derivatives.A methyl ester derivative 5a was synthesized via PCl 3 coupling.Ester hydrolysis with LiOH produced carboxylic acid analogue 6 which was intended to serve as a scaffold to produce a small library of amide analogues (Schemes 2 and 3).

Chemistry
The nitro group on niclosamide was chosen as an initial site for modification.We initially sought to synthesize two series of amide analogs, one of which linked the amide to the salicylanilide scaffold via the nitrogen and one that was linked via the carbonyl group.The first series of derivatives resulted from treating niclosamide with zinc dust in the presence of NH4Cl, yielding primary amine derivative 1.As an initial proof of concept, the amine was further acylated and alkylated to produce compounds 2 and 3 (Scheme 1).The other series of amide containing compounds was synthesized by coupling commercially available 5-chlorosalisylic acid with various aniline derivatives.A methyl ester derivative 5a was synthesized via PCl3 coupling.Ester hydrolysis with LiOH produced carboxylic acid analogue 6 which was intended to serve as a scaffold to produce a small library of amide analogues (Schemes 2 and 3).
Initial attempts to derivatize acid 6 via amide coupling proved unsuccessful with analysis revealing the phenol in niclosamide posing synthetic challenges.Two phenol protecting group strategies were developed to overcome this challenge: a methyl ether protecting group that could be deprotected using BBr3 and a benzyl protecting group that could be deprotected with catalytic hydrogenolysis.The role of the phenol functional group was also explored using intermediate 5b, analogous to 5a but with a methyl ether protected phenol.These protecting group strategies enabled the development of amide derivatives 7-12 which allowed for the introduction of a variety of functional groups (Scheme 3).Initial attempts to derivatize acid 6 via amide coupling proved unsuccessful with analysis revealing the phenol in niclosamide posing synthetic challenges.Two phenol protecting group strategies were developed to overcome this challenge: a methyl ether protecting group that could be deprotected using BBr 3 and a benzyl protecting group that could be deprotected with catalytic hydrogenolysis.The role of the phenol functional group was also explored using intermediate 5b, analogous to 5a but with a methyl ether protected phenol.These protecting group strategies enabled the development of amide derivatives 7-12 which allowed for the introduction of a variety of functional groups (Scheme 3).An azide derivative 4 was also prepared by reacting 5-chlorosalisylic acid with 4azidoaniline in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).Attempts to synthesize an ethynylaniline derivative with an alkyne at the same position as the azide group proved unsuccessful, so an alternative strategy to install the alkyne group was utilized.Compound 13, a propargylamide derivative, was coupled to various azide-containing fragments via copper-assisted azide-alkyne cycloaddition (CuAAC) [16] in Scheme 4 to explore the effects of a triazole moiety in compounds 14-17.An azide derivative 4 was also prepared by reacting 5-chlorosalisylic acid with 4azidoaniline in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).Attempts to synthesize an ethynylaniline derivative with an alkyne at the same position as the azide group proved unsuccessful, so an alternative strategy to install the alkyne group was utilized.Compound 13, a propargylamide derivative, was coupled to various azide-containing fragments via copper-assisted azide-alkyne cycloaddition (CuAAC) [16] in Scheme 4 to explore the effects of a triazole moiety in compounds 14-17.
Lastly, a series of polyphenol derivatives were synthesized (Scheme 5).Since the phenol group on niclosamide was previously shown to be essential for its activity as a protonophore [17] and thus its synergy with colistin [13], we wanted to explore the effect of installing additional phenol groups.The salicylic acid ring of niclosamide was modified with additional hydroxyl groups (18,19) and the previously described amide coupling method was used to produce polyphenol analogs 20 and 21.
Attempts to synthesize an ethynylaniline derivative with an alkyne at the same position as the azide group proved unsuccessful, so an alternative strategy to install the alkyne group was utilized.Compound 13, a propargylamide derivative, was coupled to various azide-containing fragments via copper-assisted azide-alkyne cycloaddition (CuAAC) [16] in Scheme 4 to explore the effects of a triazole moiety in compounds 14-17.Lastly, a series of polyphenol derivatives were synthesized (Scheme 5).Since the phenol group on niclosamide was previously shown to be essential for its activity as a protonophore [17] and thus its synergy with colistin [13], we wanted to explore the effect of installing additional phenol groups.The salicylic acid ring of niclosamide was modified with additional hydroxyl groups (18,19) and the previously described amide coupling method was used to produce polyphenol analogs 20 and 21.

Initial Screen for Synergy with Colistin and SAR
The initial screen of our study aimed to evaluate the ability of compounds 1-21 to synergize with colistin.For the initial screen, two colistin-resistant clinical isolates of Klebsiella pneumoniae (KP113250 and KP113254) and Escherichia coli (EC94393 and EC94474) were chosen.The two K. pneumoniae strains were chosen as they are highly colistin-resistant with minimum inhibitory concentrations (MIC) for colistin of 256 µg/mL.The two E. coli strains were selected as they were known to harbor the mcr-1 gene [18] which confers colistin resistance via phosphoethanolamine transferase [19].Colistin had MICs of 8 µg/mL in EC94394 and 16 µg/mL in EC94474.
To ensure any biological activity seen was due to colistin potentiation and not to any innate activity of the newly synthesized compounds, we first screened all compounds for standalone antibacterial activity against these four strains (Table S1).The lowest concentration of each compound able to inhibit bacterial growth was determined to be the MIC.Similar to niclosamide, all compounds displayed poor antibacterial activity with MIC values of ≥128 µg/mL and were then further evaluated for synergy with colistin.
To perform the initial screen, we used a fixed concentration of 4 µM of compound and determined the MIC of colistin in combination against each strain.As a comparison, niclosamide was shown to drastically enhance the antibacterial activity of colistin in all

Biological Evaluations 2.2.1. Initial Screen for Synergy with Colistin and SAR
The initial screen of our study aimed to evaluate the ability of compounds 1-21 to synergize with colistin.For the initial screen, two colistin-resistant clinical isolates of Klebsiella pneumoniae (KP113250 and KP113254) and Escherichia coli (EC94393 and EC94474) were chosen.The two K. pneumoniae strains were chosen as they are highly colistin-resistant with minimum inhibitory concentrations (MIC) for colistin of 256 µg/mL.The two E. coli strains were selected as they were known to harbor the mcr-1 gene [18] which confers colistin resistance via phosphoethanolamine transferase [19].Colistin had MICs of 8 µg/mL in EC94394 and 16 µg/mL in EC94474.
To ensure any biological activity seen was due to colistin potentiation and not to any innate activity of the newly synthesized compounds, we first screened all compounds for standalone antibacterial activity against these four strains (Table S1).The lowest concentration of each compound able to inhibit bacterial growth was determined to be the MIC.Similar to niclosamide, all compounds displayed poor antibacterial activity with MIC values of ≥128 µg/mL and were then further evaluated for synergy with colistin.
To perform the initial screen, we used a fixed concentration of 4 µM of compound and determined the MIC of colistin in combination against each strain.As a comparison, niclosamide was shown to drastically enhance the antibacterial activity of colistin in all four strains, consistent with previous reports [9].At 4 µM, niclosamide was able to lower the MIC of colistin by 512-fold against KP113250, 1024-fold against KP113254, and 32-fold against both E. coli strains.The prominent colistin MIC reduction by niclosamide provided a baseline of potentiating activity to which the new derivatives could be compared.At first, we investigated colistin potentiation for compounds 1-3 at 4 µM (Table 1).We observed that the conversion of the nitro group to an amine moiety (1) resulted in comparable potentiation of colistin in E. coli strains.However, reduced potentiation was observed against K. pneumoniae.Further modifying the amine with acylation (2) or alkylation (3) led to a complete loss of colistin-potentiating activity against both bacteria (Table 1).
Antibiotics 2024, 13, x FOR PEER REVIEW against K. pneumoniae.Further modifying the amine with acylation (2) or alkylation to a complete loss of colistin-potentiating activity against both bacteria (Table 1).We next replaced the nitro group on niclosamide with an azide (4), a methy (5a), a carboxylic acid (6), various amide moieties (7)(8)(9)(10)(11)(12), and triazole fragments ( (Table 2).We observed that the azide and methyl ester derivatives retained synerg colistin against all strains tested, indicating that these modifications are compatibl the colistin potentiation of niclosamide.Compound 4 displayed prominent poten activity against the two E. coli strains, reducing the MIC of colistin to 0.016 µg/mL a EC94393 (a 16-fold higher reduction compared to niclosamide).As 4 also contains h gen at R 1 instead of a chlorine atom, it appears that the chlorine at this position necessary for synergy with colistin.Compound 5a, in combination with colistin, disp a comparable synergy profile relative to niclosamide in all tested strains.Interes compound 5b, which differs from 5a only by a methylated phenol group, lost all sy with colistin (Table S2).This demonstrates the necessity of the phenol in niclosam ability to potentiate colistin.Unfortunately, further modification of the methyl este ety by converting it to a carboxylic acid (6) or an amide (7-12) completely abolish synergy with colistin.The triazole analogs 14-17 were not able to potentiate colistin tested strains.Nonetheless, these findings demonstrated that the nitro group on n mide can be replaced with a variety of substituents while still retaining synergy wi istin.We next replaced the nitro group on niclosamide with an azide (4) (5a), a carboxylic acid (6), various amide moieties (7)(8)(9)(10)(11)(12), and triazole fra (Table 2).We observed that the azide and methyl ester derivatives retaine colistin against all strains tested, indicating that these modifications are c the colistin potentiation of niclosamide.Compound 4 displayed promine activity against the two E. coli strains, reducing the MIC of colistin to 0.016 EC94393 (a 16-fold higher reduction compared to niclosamide).As 4 also gen at R 1 instead of a chlorine atom, it appears that the chlorine at this necessary for synergy with colistin.Compound 5a, in combination with co a comparable synergy profile relative to niclosamide in all tested strain compound 5b, which differs from 5a only by a methylated phenol group, with colistin (Table S2).This demonstrates the necessity of the phenol i ability to potentiate colistin.Unfortunately, further modification of the m ety by converting it to a carboxylic acid (6) or an amide (7-12) complete synergy with colistin.The triazole analogs 14-17 were not able to potentia tested strains.Nonetheless, these findings demonstrated that the nitro gr mide can be replaced with a variety of substituents while still retaining sy istin.We next replaced the nitro group on niclosamide with an azide (4) (5a), a carboxylic acid (6), various amide moieties (7)(8)(9)(10)(11)(12), and triazole fra (Table 2).We observed that the azide and methyl ester derivatives retaine colistin against all strains tested, indicating that these modifications are c the colistin potentiation of niclosamide.Compound 4 displayed promine activity against the two E. coli strains, reducing the MIC of colistin to 0.016 EC94393 (a 16-fold higher reduction compared to niclosamide).As 4 also gen at R 1 instead of a chlorine atom, it appears that the chlorine at this necessary for synergy with colistin.Compound 5a, in combination with col a comparable synergy profile relative to niclosamide in all tested strain compound 5b, which differs from 5a only by a methylated phenol group, with colistin (Table S2).This demonstrates the necessity of the phenol i ability to potentiate colistin.Unfortunately, further modification of the m ety by converting it to a carboxylic acid (6) or an amide (7-12) complete synergy with colistin.The triazole analogs 14-17 were not able to potentia tested strains.Nonetheless, these findings demonstrated that the nitro gr mide can be replaced with a variety of substituents while still retaining sy istin.We next replaced the nitro group on niclosamide with an azide (4), a methyl ester (5a), a carboxylic acid (6), various amide moieties (7)(8)(9)(10)(11)(12), and triazole fragments (14-17) (Table 2).We observed that the azide and methyl ester derivatives retained synergy with colistin against all strains tested, indicating that these modifications are compatible with the colistin potentiation of niclosamide.Compound 4 displayed prominent potentiating activity against the two E. coli strains, reducing the MIC of colistin to 0.016 µg/mL against EC94393 (a 16-fold higher reduction compared to niclosamide).As 4 also contains hydrogen at R 1 instead of a chlorine atom, it appears that the chlorine at this position is not necessary for synergy with colistin.Compound 5a, in combination with colistin, displayed a comparable synergy profile relative to niclosamide in all tested strains.Interestingly, compound 5b, which differs from 5a only by a methylated phenol group, lost all synergy with colistin (Table S2).This demonstrates the necessity of the phenol in niclosamide's ability to potentiate colistin.Unfortunately, further modification of the methyl ester moiety by converting it to a carboxylic acid (6) or an amide (7)(8)(9)(10)(11)(12) completely abolished all synergy with colistin.The triazole analogs 14-17 were not able to potentiate colistin in all tested strains.Nonetheless, these findings demonstrated that the nitro group on niclosamide can be replaced with a variety of substituents while still retaining synergy with colistin.ability to potentiate colistin.Unfortunately, further modification of the methyl este ety by converting it to a carboxylic acid (6) or an amide (7-12) completely abolish synergy with colistin.The triazole analogs 14-17 were not able to potentiate colistin tested strains.Nonetheless, these findings demonstrated that the nitro group on n mide can be replaced with a variety of substituents while still retaining synergy wi istin.Given the demonstrated importance of the phenol moiety within the salicyl scaffold, we wondered how the installation of additional phenol groups, as in comp 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) resu a complete loss of synergy with colistin.The amide-containing polyphenol compou and 21 were also inactive.These results suggest that while the phenol group is nec the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicyl scaffold, we wondered how the installation of additional phenol groups, as in comp 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) resu a complete loss of synergy with colistin.The amide-containing polyphenol compou and 21 were also inactive.These results suggest that while the phenol group is nec the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicyl scaffold, we wondered how the installation of additional phenol groups, as in comp 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) resu a complete loss of synergy with colistin.The amide-containing polyphenol compou and 21 were also inactive.These results suggest that while the phenol group is nec the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicyl scaffold, we wondered how the installation of additional phenol groups, as in comp 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) resu a complete loss of synergy with colistin.The amide-containing polyphenol compou and 21 were also inactive.These results suggest that while the phenol group is nec the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicy scaffold, we wondered how the installation of additional phenol groups, as in com 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) res a complete loss of synergy with colistin.The amide-containing polyphenol compo and 21 were also inactive.These results suggest that while the phenol group is ne the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicy scaffold, we wondered how the installation of additional phenol groups, as in com 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) res a complete loss of synergy with colistin.The amide-containing polyphenol compo and 21 were also inactive.These results suggest that while the phenol group is nec the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicy scaffold, we wondered how the installation of additional phenol groups, as in com 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) res a complete loss of synergy with colistin.The amide-containing polyphenol compo and 21 were also inactive.These results suggest that while the phenol group is ne the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicyl scaffold, we wondered how the installation of additional phenol groups, as in comp 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We observ the addition of even one additional phenol group on the salicylic acid ring (18) resu a complete loss of synergy with colistin.The amide-containing polyphenol compou and 21 were also inactive.These results suggest that while the phenol group is nec the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicy scaffold, we wondered how the installation of additional phenol groups, as in com 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We obser the addition of even one additional phenol group on the salicylic acid ring (18) res a complete loss of synergy with colistin.The amide-containing polyphenol compo and 21 were also inactive.These results suggest that while the phenol group is ne the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the sali scaffold, we wondered how the installation of additional phenol groups, as in co 18-21, would affect niclosamide s ability to potentiate colistin (Table 3).We obs the addition of even one additional phenol group on the salicylic acid ring (18) r a complete loss of synergy with colistin.The amide-containing polyphenol comp and 21 were also inactive.These results suggest that while the phenol group is the addition of multiple phenol groups reduces colistin-potentiating activity.Given the demonstrated importance of the phenol moiety within the salicylanilide scaffold, we wondered how the installation of additional phenol groups, as in compounds 18-21, would affect niclosamide's ability to potentiate colistin (Table 3).We observed that the addition of even one additional phenol group on the salicylic acid ring (18) resulted in a complete loss of synergy with colistin.The amide-containing polyphenol compounds 20 and 21 were also inactive.These results suggest that while the phenol group is necessary, the addition of multiple phenol groups reduces colistin-potentiating activity.Overall, the results of this SAR led to us conclude that the nitro group in niclosamide can be replaced with an azide, a methyl ester, and (to a lesser extent) an amine while still retaining synergy with colistin against GNB.We also show that further modification of the amine or ester groups abolishes synergistic activity.The phenol group was shown to be necessary for synergy with colistin as methylation resulted in complete loss of colistin potentiation (compound 5b, Table S2).
Finally, we compared the colistin potentiation of hit compounds 1, 4 and 5a to niclosamide in a panel of colistin-resistant and colistin-susceptible GNB using the checkerboard assay (Figure 2).The fractional inhibitory concentration index (FICI) for each combination was determined to assess interactions between the two components.FICI values of ≤0.5, 0.5 < x ≤ 4, and >4 were interpreted as synergistic, additive, and antagonistic interactions, respectively.We observed that compound 4 was overall superior to niclosamide (lowest FICI value), especially in E. coli.Compound 5a was comparable to niclosamide, while compound 1 showed synergy with colistin only in E. coli and K. pneumoniae but not in Pseudomonas aeruginosa.Despite the promising colistin-potentiating activity of 4, the high synthetic cost as well as concerns over the stability of the aryl azide led to us selecting the methyl ester compound 5a for further screening against clinical isolates of P. aeruginosa and Acinetobacter baumannii.Overall, the results of this SAR led to us conclude that the nitro group in niclosamide can be replaced with an azide, a methyl ester, and (to a lesser extent) an amine while still retaining synergy with colistin against GNB.We also show that further modification of the amine or ester groups abolishes synergistic activity.The phenol group was shown to be necessary for synergy with colistin as methylation resulted in complete loss of colistin potentiation (compound 5b, Table S2).
Finally, we compared the colistin potentiation of hit compounds 1, 4 and 5a to niclosamide in a panel of colistin-resistant and colistin-susceptible GNB using the checkerboard assay (Figure 2).The fractional inhibitory concentration index (FICI) for each combination was determined to assess interactions between the two components.FICI values of ≤0.5, 0.5 < x ≤ 4, and >4 were interpreted as synergistic, additive, and antagonistic interactions, respectively.We observed that compound 4 was overall superior to niclosamide (lowest FICI value), especially in E. coli.Compound 5a was comparable to niclosamide, while compound 1 showed synergy with colistin only in E. coli and K. pneumoniae but not in Pseudomonas aeruginosa.Despite the promising colistin-potentiating activity of 4, the high synthetic cost as well as concerns over the stability of the aryl azide led to us selecting the methyl ester compound 5a for further screening against clinical isolates of P. aeruginosa and Acinetobacter baumannii.Overall, the results of this SAR led to us conclude that the nitro group in niclosamide can be replaced with an azide, a methyl ester, and (to a lesser extent) an amine while still retaining synergy with colistin against GNB.We also show that further modification of the amine or ester groups abolishes synergistic activity.The phenol group was shown to be necessary for synergy with colistin as methylation resulted in complete loss of colistin potentiation (compound 5b, Table S2).
Finally, we compared the colistin potentiation of hit compounds 1, 4 and 5a to niclosamide in a panel of colistin-resistant and colistin-susceptible GNB using the checkerboard assay (Figure 2).The fractional inhibitory concentration index (FICI) for each combination was determined to assess interactions between the two components.FICI values of ≤0.5, 0.5 < x ≤ 4, and >4 were interpreted as synergistic, additive, and antagonistic interactions, respectively.We observed that compound 4 was overall superior to niclosamide (lowest FICI value), especially in E. coli.Compound 5a was comparable to niclosamide, while compound 1 showed synergy with colistin only in E. coli and K. pneumoniae but not in Pseudomonas aeruginosa.Despite the promising colistin-potentiating activity of 4, the high synthetic cost as well as concerns over the stability of the aryl azide led to us selecting the methyl ester compound 5a for further screening against clinical isolates of P. aeruginosa and Acinetobacter baumannii.Overall, the results of this SAR led to us conclude that the nitro group in niclosamide can be replaced with an azide, a methyl ester, and (to a lesser extent) an amine while still retaining synergy with colistin against GNB.We also show that further modification of the amine or ester groups abolishes synergistic activity.The phenol group was shown to be necessary for synergy with colistin as methylation resulted in complete loss of colistin potentiation (compound 5b, Table S2).
Finally, we compared the colistin potentiation of hit compounds 1, 4 and 5a to niclosamide in a panel of colistin-resistant and colistin-susceptible GNB using the checkerboard assay (Figure 2).The fractional inhibitory concentration index (FICI) for each combination was determined to assess interactions between the two components.FICI values of ≤0.5, 0.5 < x ≤ 4, and >4 were interpreted as synergistic, additive, and antagonistic interactions, respectively.We observed that compound 4 was overall superior to niclosamide (lowest FICI value), especially in E. coli.Compound 5a was comparable to niclosamide, while compound 1 showed synergy with colistin only in E. coli and K. pneumoniae but not in Pseudomonas aeruginosa.Despite the promising colistin-potentiating activity of 4, the high synthetic cost as well as concerns over the stability of the aryl azide led to us selecting the methyl ester compound 5a for further screening against clinical isolates of P. aeruginosa and Acinetobacter baumannii.

Synergy between 5a and Colistin against P. aeruginosa and A. baumannii
We further evaluated the therapeutic potential of lead compound 5a in combination with colistin against a panel of multidrug-resistant (MDR) clinical isolates of P. aeruginosa and A. baumannii (Tables 4 and 5).Compound 5a was able to synergize with colistin in 8 out of 10 strains of P. aeruginosa.Synergy was only not observed in PA259 and PA260, which were already highly susceptible to colistin (MIC of 0.25 µg/mL).Analog 5a was able to drastically reduce colistin's MIC against all colistin-resistant isolates (PA91433, PA101243, PA262 and PA101885).The combination of 5a and colistin against PA101243 was of particular note, with 1 µg/mL of 5a lowering the MIC of colistin from 1024 µg/mL to

Synergy between 5a and Colistin against P. aeruginosa and A. baumannii
We further evaluated the therapeutic potential of lead compound 5a in combination with colistin against a panel of multidrug-resistant (MDR) clinical isolates of P. aeruginosa and A. baumannii (Tables 4 and 5).Compound 5a was able to synergize with colistin in 8 out of 10 strains of P. aeruginosa.Synergy was only not observed in PA259 and PA260, which were already highly susceptible to colistin (MIC of 0.25 µg/mL).Analog 5a was able to drastically reduce colistin s MIC against all colistin-resistant isolates (PA91433, PA101243, PA262 and PA101885).The combination of 5a and colistin against PA101243 was of particular note, with 1 µg/mL of 5a lowering the MIC of colistin from 1024 µg/mL to 0.5 µg/mL, a 2048-fold reduction.In all strains tested, 5a was able to lower the MIC of colistin to 0.5 µg/mL or less (Table 4).Similar synergistic activity was observed for A. baumannii (Table 5).Synergy was observed in four out of five isolates, LAC-4 (colistin MIC of 0.25 µg/mL) being the only exception.Compound 5a was able to lower the MIC of colistin to 0.5 µg/mL or below in all strains tested, including against highly resistant AB027 (colistin MIC of 1024 µg/mL).
Overall, these results show that a combination of colistin and 5a is effective against K. pneumonia, E. coli, P. aeruginosa and A. baumannii.Replacing the nitro group of niclosamide with a methyl ester led to the development of a new derivative that synergized with colistin in nearly all clinical isolates tested, and restored colistin activity against every colistin-resistant isolate.

Cytotoxicity of 1 and 5a against Eukaryotic Cells
Toxicity against eukaryotic cells is a major challenge for repurposing niclosamide as an antibacterial agent.To test whether the modification of the nitro group may be a potential avenue for reducing toxicity, the toxic effects of compounds 1 and 5a were investigated on two ovarian cancer cell lines OVCAR-3 and COV362, and compared to niclosamide which has been shown to possess potent antitumor effects against ovarian cancer cells [20].The results of experiments with the three compounds using the CyQuant assay [21] are displayed in Figure 3.After 48 h incubation, the cell number for the controls increased by about 26% for OVCAR-3 and 89% for COV362 relative to the initial cell number (day 0).Overall, these results show that a combination of colistin and 5a is effective against K. pneumonia, E. coli, P. aeruginosa and A. baumannii.Replacing the nitro group of niclosamide with a methyl ester led to the development of a new derivative that synergized with colistin in nearly all clinical isolates tested, and restored colistin activity against every colistin-resistant isolate.

Cytotoxicity of 1 and 5a against Eukaryotic Cells
Toxicity against eukaryotic cells is a major challenge for repurposing niclosamide as an antibacterial agent.To test whether the modification of the nitro group may be a potential avenue for reducing toxicity, the toxic effects of compounds 1 and 5a were investigated on two ovarian cancer cell lines OVCAR-3 and COV362, and compared to niclosamide which has been shown to possess potent antitumor effects against ovarian cancer cells [20].The results of experiments with the three compounds using the CyQuant assay [21] are displayed in Figure 3.After 48 h incubation, the cell number for the controls increased by about 26% for OVCAR-3 and 89% for COV362 relative to the initial cell number (day 0).Cell numbers from representative wells were determined on day 0 before the addition of the compound.After 48 h incubation (day 2), cells were counted again to determine the increase in cell number over day 0 cell count.The overall increase in cell number for control after 48 h incubation was found to be around 26% and 89% relative to the initial Figure 3.Effect of niclosamide, compound 1 and compound 5a (0-100 µM) on ovarian cancer cell lines (a) OVCAR-3 and (b) COV362 cells.Cell numbers from representative wells were determined on day 0 before the addition of the compound.After 48 h incubation (day 2), cells were counted again to determine the increase in cell number over day 0 cell count.The overall increase in cell number for control after 48 h incubation was found to be around 26% and 89% relative to the initial cell number (day 0) for OVCAR-3 and COV362 cells, respectively.Each concentration of the drug tested is represented as a percent of that cell growth relative to the initial cell number.The results represent the mean ± standard deviation of two independent experiments with four wells for each concentration.
Incubation with 6-100 µM niclosamide after 48 h showed a toxic effect on both the cell lines, killing around 60% (OVCAR-3) and 30% (COV362) of the initial cell number before drug treatment.Incubation of cells with compound 5a at 6-50 µM killed only 20% of OVCAR-3 cells while it had a cytostatic effect (no significant change in initial cell number) in the same concentration range in COV362 cells.On the other hand, compound 1 at 6-50 µM showed slight inhibition of proliferation (around 13%) in OVCAR-3 cells relative to the control whereas under the same incubation conditions at 6-25 µM in COV362 cells, it stimulated proliferation and increased the cell number by 14% more than the control.Thus, in both OVCAR-3 and COV362 cells, incubation with compound 1 resulted in cell numbers higher than the initial cell number indicating a non-cytotoxic effect in the range of 6-25 µM.
Compound 5a and compound 1 at 100 µM killed 43% and 15% of OVCAR-3 cells, respectively.At the same concentration, in COV362 cells, compound 5a and compound 1 showed 75% and 66% inhibition of proliferation relative to the control but the cell numbers were still higher than the initial cell number indicating an antiproliferative rather than a cytotoxic effect.Taken together, the results demonstrate that compounds 1 and 5a are less cytotoxic than niclosamide at all concentrations tested.Since the results of the microbiological studies demonstrate that compound 5a was able to synergize with colistin to eradicate colistin-resistant GNB strains tested at concentrations of 1µg/mL (~0.3 µM), these results are very promising as an initial demonstration of reduced cytotoxicity.Overall, these results show that the replacement of the nitro group in niclosamide is a viable strategy to reduce toxicity against eukaryotic cells while retaining synergy with colistin.

Materials
Reagents and solvents were purchased from commercially available suppliers such as Sigma-Aldrich (St Louis, MO, USA), AK Scientific (Union City, CA, USA), Fisher Scientific (Waltham, MA, USA), and Bachem (Bubendorf, Switzerland) and were used without further purification.Air-and moisture-sensitive reactions were performed with dry solvents under inert nitrogen atmosphere.The reaction progress was monitored using thin-layer chromatography (TLC) plates visualized with UV light.Column chromatography was performed to purify the compounds using SiliaFlash P60 (40-63 µm) 60 Å silica gel from Silicycle (Quebec City, QC, Canada).The chemical structures of all final products used for biological testing were characterized by nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR) on a Bruker AMX NMR 300 MHz and 500 MHz spectrometer (Bruker, Billerica, MA, USA).Electrospray ionization mass spectrometry (ESI-MS) data were obtained using a Varian 320-MS LC/MS (Agilent, Santa Clara, CA, USA).

General Synthetic Procedures
General procedure A: Benzanilide coupling using PCl 3 The appropriate benzoic acid derivative (1 equiv.)and aniline derivative (1 equiv.)were dissolved in dry xylene (2 mL per mmol of reactants) and heated to reflux.PCl 3 (0.4 equiv.) was added dropwise upon which a precipitate formed.Solution was refluxed for 4 h.The mixture was cooled to rt and filtered, after which the precipitate was washed with ethyl acetate.
General procedure B: LiOH Ester hydrolysis The appropriate carboxylic acid was dissolved in a THF (5 mL per mmol) and MeOH (5 mL per mmol) mixture.A solution of 2M LiOH in water (10 mL per mmol) was added and the reaction was stirred at rt for 2 h.The reaction was neutralized with concentrated HCl at 0 • C upon which the product precipitated, was filtered and washed with water (3 × 10 mL).
General procedure D: Methoxy deprotection The appropriate methoxy protected intermediate (1 equiv.) was dissolved in anhydrous DCM at 0 • C. A 1M solution of BBr 3 in DCM (1.5 equiv.BBr 3 per methoxy group) was added dropwise and the reaction was slowly warmed to rt.Solution was stirred overnight.
The reaction was cooled to 0 • C and quenched with the careful addition of MeOH (5 mL per mmol) followed by stirring for 15 min.The reaction mixture was concentrated in vacuo and the product was purified using flash chromatography (0-10% MeOH in DCM).
General procedure E: Benzyl deprotection The appropriate benzyl protected intermediate (1 equiv.) was dissolved in MeOH (3 mL per mmol) and THF (1 mL per mmol) Palladium on carbon (0.1 equiv.) was then added followed by H 2 gas (balloon).The solution was stirred for 2 h at rt, filtered over celite and concentrated in vacuo.The product was purified using flash chromatography (0-10% MeOH in DCM).
General procedure F: Copper-assisted azide-alkyne cycloaddition (CuAAC) The appropriate azide (1 equiv.)and alkyne (1 equiv.)and CuI•P(OEt) 3 (0.3 equiv.)were dissolved in anhydrous DMF (3 mL) and treated with iPr 2 NEt (2 equiv.)The reaction was stirred under N 2 at rt for 2 h.The reaction mixture was concentrated under vacuum, dissolved in water and extracted with DCM (× 3) The organic layers were combined, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo.The product was purified using flash chromatography (0-10% MeOH in DCM).

Antimicrobial Susceptibility Assay
Bacterial samples for this research were sourced from the American Type Culture Collection (ATCC), the Canadian National Intensive Care Unit (CAN-ICU) surveillance study [22], and the Canadian Ward (CANWARD) surveillance study [23].Clinical samples from the CAN-ICU and CANWARD studies were taken from patients with suspected infectious diseases in participating medical centers across Canada during the study period.The antibacterial properties of the compounds were evaluated using the microbroth dilution technique as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [24].Bacterial cultures were cultured overnight and then diluted in saline to a 0.5 McFarland standard.This was further diluted 1:50 in Cation-adjusted Mueller-Hinton broth (CAMHB) to yield a concentration of about 5 × 10 5 CFU/mL.Tests were conducted in 96-well plates.The agents were serially diluted in CAMHB and incubated with bacterial samples at 37 • C for 18 h.The minimum inhibitory concentration (MIC) was determined as the lowest concentration preventing visible bacterial growth, confirmed using an EMax Plus microplate reader (Molecular Devices, San Jose, CA, USA) at 590 nm.CAMHB, with or without bacterial cells, served as positive and negative controls, respectively.

Checkerboard Assay
The experiment was conducted in 96-well plates as described previously [25].One agent underwent a 2-fold serial dilution along the x-axis, while the other was similarly diluted along the y-axis, resulting in a matrix where each well held both agents at varying concentrations.Bacterial cultures, cultivated overnight, were diluted in saline to achieve a 0.5 McFarland turbidity.This was then further diluted 1:50 in CAMHB, and each well was inoculated, reaching an approximate concentration of 5 × 10 5 CFU/mL.Wells containing only CAMHB, with or without bacteria, served as the positive and negative controls, respectively.The plates were incubated at 37 • C for 18 h and checked for visible turbidity, verified with an EMax Plus microplate reader (Molecular Devices, USA) at 590 nm.The fractional inhibitory concentration (FIC) for each agent was determined by dividing the MIC of the compound with colistin by its MIC when alone.Similarly, the FIC for colistin was determined by dividing its MIC with each compound by its standalone MIC.The FIC index was derived by adding both FIC values together.FIC indices were classified as synergistic (≤0.5), indifferent (0.5 < x ≤ 4), or antagonistic (>4).

Cell Proliferation Assay
Cell proliferation was assessed by quantifying changes in cell numbers using the CyQuant cell proliferation assay (Invitrogen, Waltham, MA, USA), essentially as described by the manufacturer.OVCAR-3 and COV362 cells were seeded in 96-well plates (7500 cells/well) in a volume of 100 µL.Wells with only media and no cells served as blanks.The plates were maintained in a 5% CO 2 incubator at 37 • C. Two additional plates with cells and blank wells were prepared as control plates (did not receive the drug treatment) for determination of the cell numbers on the day of the addition of the drugs (day 0) and after the 48 h incubation absolute cell numbers from representative wells from these plates were determined with a Coulter ZM counter, while the remainder wells were processed for the CyQuant assay as described below.When cells were in the log phase, drug solutions were added to the test plates to yield final concentrations of 0-100 µM.The control plates received the media with the vehicle.After 48 h incubation, the media was removed, and the plates were placed at −80 • C for 7 days.The plates were allowed to thaw to room temperature, and CyQuant reagent in lysis buffer (200 µL) was added to each well.Fluorescence was measured on a SpectraMax M2 microplate reader (Molecular Devices, USA) at excitation and emission wavelengths of 480 and 520 nm, respectively.The results were expressed as percent cell number relative to initial cell number for each concentration.

Conclusions
A series of niclosamide analogs were synthesized to perform an SAR with an overall goal of replacing the nitro group of niclosamide to reduce toxicity while maintaining the ability of niclosamide to synergize with colistin against GNB.A small library of compounds was produced and their ability to synergize with colistin was observed.It was found that the nitro group on niclosamide can be replaced by an amine, a methyl ester, or an azide while still retaining colistin-potentiating activity.We also showed that the phenol group of niclosamide was necessary for synergy with colistin, but that the addition of multiple phenols led to a complete loss of synergy.The methyl ester analog (5a) was assessed against a panel of MDR clinical isolates and was shown to reduce colistin MIC to below CLSI breakpoint values, even against highly resistant isolates.The amine and methyl ester compounds were also found to be less toxic to eukaryotic cells.One significant challenge that needs to be overcome is the likely metabolic instability of the ester in compound 5a, as esters are known to be prone to hydrolysis in vivo [26], especially given that the carboxylic acid derivative 6 displayed no synergy with colistin.Nonetheless, this work provides important insights into synthetic strategies for the future development of new niclosamide derivatives, and that modification to the nitro group of niclosamide may be a viable strategy of reducing toxicity.

Figure 1 .
Figure 1.Structures of niclosamide, rafoxanide, and proposed new scaffolds.The initial site of modification is highlighted.

Figure 1 .
Figure 1.Structures of niclosamide, rafoxanide, and proposed new scaffolds.The initial site of modification is highlighted.

Table 2 .2
Colistin MIC in combination with 4 µM of compounds 4-12 and 14-17 against four c resistant strains of GNB.KP = Klebsiella pneumoniae, EC = Escherichia coli.Colistin MIC with 4 µM Compound (µg/mL) Antibiotics 2024, 13, x FOR PEER REVIEW against K. pneumoniae.Further modifying the amine with acylation (2) or a to a complete loss of colistin-potentiating activity against both bacteria (Ta

Table 2 .
Colistin MIC in combination with 4 µM of compounds 4-12 and 14-17 aga resistant strains of GNB.KP = Klebsiella pneumoniae, EC = Escherichia coli.Antibiotics 2024, 13, x FOR PEER REVIEW against K. pneumoniae.Further modifying the amine with acylation (2) or a to a complete loss of colistin-potentiating activity against both bacteria (Ta

Table 4 .
Synergy between compound 5a and colistin against a panel of wild-type and clinical isolates of P. aeruginosa.

Table 4 .
Synergy between compound 5a and colistin against a panel of wild-type and clinical isolates of P. aeruginosa.

Table 5 .
Synergy between compound 5a and colistin against a panel of wild-type and clinical isolates of A. baumannii.